Complete Depletion of Primordial Germ Cells Results in Masculinization of Monopterus albus, a Protogynous Hermaphroditic Fish.

Primordial germ cells (PGCs) play an important role in sexual fate determination and gonadal development in gonochoristic fish, such as zebrafish and medaka. However, little is known about the function of PGCs in hermaphroditic fish. Rice field eel (Monopterus albus), a protogynous hermaphroditic fish, is an economically valuable aquaculture species. We eliminated PGCs in rice field eels during embryogenesis via morpholino-mediated knockdown dead end (dnd). The PGCs-depleted gonads developed into testis-like structures with Sertoli cells and Leydig cells. The gene expression pattern of 15-month-old PGCs-depleted gonads showed that male-biased genes, dmrt1, sox9a, gsdf, and amh, were significantly higher than that of the WT, whereas female-biased genes, foxl2 and cyp19a1a, were significantly decreased. These results indicate that PGCs are essential for ovarian differentiation in rice field eel, and PGCs-depleted gonads develop into sterile males without undergoing the female and intersex stages. Our study is the first to identify the role of PGCs in sex differentiation in rice field eel, a protogynous hermaphrodite teleost. And it is of great significance in rice field eel for discovering the underlying mechanism of sex differentiation and establishing sex control technology.

Identification and molecular characterization of peroxiredoxin 2 in grass carp (Ctenopharyngodon idella).

Peroxiredoxin (Prx), also named thioredoxin peroxidase (TPx), is a selenium independent antioxidant enzyme that can protect organisms from oxidative damage caused by reactive oxygen species (ROS) and is important for immune responses. In this study, the molecular cloning and characterization of a Prx2 homologue (CiPrx2) were described from grass carp (Ctenopharyngodon idella). The full-length cDNA of CiPrx2 was 1163 bp containing 5'-untranslated region (UTR) of 52 bp, a 3'-UTR of 517 bp with the putative polyadenylation consensus signal (AATAAA), an open reading frame (ORF) of 594 bp encoding polypeptides of 197 amino acids with a predicted molecular mass of 21.84 kDa and theoretical isoelectric point of 5.93. The analysis results of multiple sequence alignment and phylogenetic tree confirmed that CiPrx2 belong to the typical 2-Cys Prx subfamily. The CiPrx2 mRNA was ubiquitously expressed in all tested tissues. The temporal expression of CiPrx2 were differentially induced infected with grass carp reovirus (GCRV), polyinosinic:polycytidylic acid (poly I:C) and lipopolysaccharide (LPS) in liver and spleen. Subcellular localization of CiPrx2-GFP fusion proteins were only distributed in the cytoplasm. The purified recombinant CiPrx2 possessed an apparent antioxidant activity and could protect DNA against oxidative damage. Finally, CiPrx2 proteins could obviously inhibit H2O2 and heavy metal toxicity. However, further researches are needed to better understand the regulation of CiPrx2 under oxidative stresses.

Heterologous expression of a pleiotropic drug resistance transporter from Phytophthora sojae in yeast transporter mutants.

A system for the expression of an ATP binding cassette (ABC) transporter from the soybean pathogen Phytophthora sojae is described. Pdr1, an ABC transporter with homology to the pleiotropic drug resistance (PDR) family of transporters, was cloned by primer walking from a P. sojae genomic library. Reverse transcriptase PCR assays showed that the transcript disappeared after encystment of zoospores and was not detected in hyphal germlings in dilute salts, in hyphae growing in liquid V8 media, or in tissue extracts from infected hypocotyls. BLAST analysis of Pdr1 against the P. sojae EST database also revealed that this gene was present only in zoospore libraries. Comparison of the number of hits to Pdr1 with that of a set of housekeeping genes revealed that Pdr1 was expressed at rates two- to threefold higher than other transcripts. To test the hypothesis that Pdr1p functions as a broad substrate membrane transporter, Pdr1 was transformed into yeast mutants deficient in several drug resistance transporters. Yeast mutants transformed with Pdr1 possessed partial drug resistance against only 5 of 17 chemically distinct compounds. Thus, when expressed in yeast, this transporter has a significantly narrower substrate specificity in comparison to the yeast transporters, Pdr5p, Yorlp, and Snq2p.